In optical microscopy, various methods have been developed for the depth-resolved imaging of objects, e.g., to obtain one or more sectional images through a sample. By way of example, in confocal microscopy with confocal, point-by-point illumination and imaging, image information is gathered only in the sectional plane of interest. Disadvantages of confocal microscopy exist in particular due to the complex design of the microscope and the time-consuming way in which the image information is gathered.
Another method is structured illumination microscopy (SIM technique). In this method of wide-field microscopy, a periodic grating structure is focussed into the relevant sectional plane of the sample. By imaging the grating structure of an illumination grating into the sectional plane, a modulation of the illumination in real space is achieved, thereby overcoming the so-called “missing cone” problem of wide-field microscopy. From a plurality of intermediate images recorded in each case with a different phase position of the grating structure, an image function can be calculated which represents an image of the sectional plane in the real object (see, for example, WO 2004/038483 A1). However, conventional structured illumination microscopy has the general disadvantage that the image reconstructed from the intermediate images is an approximation, the quality of which may be limited in particular by artifacts and instabilities.
For the efficient use of illumination gratings for structured illumination, use is usually made of transmission gratings which are formed by sharply defined stripes (so-called “Ronchi Ruling”). Conventional structured illumination microscopy has the general disadvantage that a transmission grating with a relatively large period is often selected for light yield reasons. As a result, the axial resolution can be improved only to a limited extent, although the potential that artifacts will occur is relatively high, depending on the observation point spread function. Another disadvantage is that Fourier components which lie outside the fundamental frequency of the transmission grating generate disadvantageous artifacts in the conventionally calculated optical sections (see L. H. Schaefer et al. in “Journal of Microscopy”, Vol. 216, 2004, pages 165-174). It has therefore been found in practice that the quality of image reconstruction may exhibit undesirable limitations, particularly when analysing biological samples. These limitations may be expressed for example in that details of the real sample are imaged only with an insufficient resolution.
It could therefore be advantageous to provide an improved method for image reconstruction from intermediate images of an object imaged with structured illumination, by means of which the disadvantages of conventional techniques are overcome. It could also be advantageous to provide an improved imaging method for obtaining images of objects illuminated with structured illumination and a correspondingly improved imaging device.